This is a proposal for a multiparameter laser scanning confocal microscope system. The microscope system consists of a detachable confocal laser scanning unit with multi-line argon laser (488 and 514 nm), dichroic emission beam splitter, and dual photon-counting detectors; upright and inverted microscopes with interchangable infinity-corrected optics; multi- channel plate-coupled intensified CCD video camera for previewing, selecting and focussing fields with minimal photobleaching and photodamage; analytical imaging workstation including computer, software and data storage for 3-dimensional image analysis; and on-stage specimen controllers for temperature and atmospheric gases (H2O, O2, CO2). The microscope will be used to examine tissues in vivo, single living cells, fixed cells, and tissue sections which have been labeled with multiple fluorescent probes or stained with light reflective compounds. In living cells, confocal images of the parameter-specific fluorescence of each probe will be collected over time during experimental manipulations, digitized and stored. Image processing and analysis will be employed to quantitate the spatial distribution of probes with a resolution of 0.2 mum in the X-Y axes and 0.8 mum in the Z axis. The analytical imaging workstation will permit reconstruction of 3-dimensional images, generation of stereo pairs, image rotation and calculation of ion concentrations. Primary uses will be determination of the 3-dimensional distribution of specific antigens in cells labeled with fluorescent antibodies, visualization of complex neural networks, determination of lipid order and topography of cell membranes, and quantitation in 3-dimensions within living cells of ions (e.g., Ca2+ and H+) and organelles (e.g., mitochondria, lysosomes, nuclear chromatin). The equipment will be utilized by many investigators on independent projects in the Lineberger Cancer Research Center and the Departments of Biochemistry, Biology, Cell Biology & Anatomy, Dermatology, Medicine, Pharmacology and Physiology of the University of North Carolina at Chapel Hill. The confocal microscope will be a unique instrument which integrates state-of-the-art advances in optical microscopy, low-light video detection and digital image analysis. Housed in the productive environment of the Video Microscopy Facility of the Laboratories for Cell Biology, the laser scanning confocal microscope system will complement existing instrumentation for fluorescence recovery after photobleaching (FRAP) and multiparameter digitized video microscopy (MDVM) and will substantially extend our ongoing efforts in applications of digitized optical microscopy to problems of biology and medicine.